NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni­(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.

NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2 eand 2 H + under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CNmolecules. Here, we investigated the sequential assembly of the [NiFe]-cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox-inactive, and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, Xray absorption, and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

A minimal light‐driven approach was established for studying enzymatic CO2 conversion spectroscopically. The system consists of a photosensitizer Eosin Y, EDTA as a sacrificial electron donor and substrate source, and formate dehydrogenase from Rhodobacter capsulatus (RcFDH) as a biocatalyst. This simplified three‐component system provides a photo‐triggered control for in situ characterization of the entire catalytic reaction. Direct reduction of RcFDH by the photosensitizer without additional electron carriers was confirmed via UV‐Vis spectroscopy, while GC‐MS and IR spectroscopy were used to follow photoinduced CO2 generation from EDTA and its subsequent enzymatic reduction, yielding the product formate. Photo‐driven and in vitro, dye‐based CO2 reduction was inhibited by azide under a mixed (competitive‐non‐competitive) inhibition mode. IR spectroscopy reveals displacement of the competitively‐bound azide by CO2, reflecting an interaction of both with the active site cofactor. This work comprises a proof‐of‐concept for a new approach to employ light for regulating the reaction of formate dehydrogenases and other CO2 reductases.

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel–iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes.